Dr Vilma Stanisich and the
Molecular genetics laboratory
E-mail:
v.stanisich@latrobe.edu.au
Projects In Plasmid Biology
(1) Mercury-resistance transposons of the Tn5053/502-family
Our interests include the epidemiology of these elements, their evolutionary relationships to
each other and to Tn (or In) elements that contain related mer (HgII-resistance) and/or tni
(transposition) modules, and the mechanistic basis of their unusual target-specificity and
their ability (under some circumstances) to transpose randomly.
(2) Evidence of evolutionary divergence, and of conservation, in promiscuous (IncPβ) plasmids
A detailed study of several IncPβ plasmids has revealed that their conserved "backbone"
has been disrupted at two locations by multiple insertions of Tn or In elements. The extant
"nested transposons" are not self-mobile, but can be induced to relocate when an appropriate
tnpA (transposase) in provided in trans. The varying molecular composition of the moveable
elements highlights the fact that seemingly "dead" transposons can continue to contribute to
bacterial diversity in unexpected ways.
Projects In Agrobacterium Biology
(3) The molecular biology of EPS (extracellular polysaccharide) production
Our studies have dealt mainly with the production of curdlan by a high-yielding
Agrobacterium strain like those used in commercial curdlan production.
This water-insoluble EPS is produced under N-depleted conditions, is structurally simple
(a linear, (1-->3)-β-glucan) and its production involves three structural genes
(crdASC) whose precise roles and regulation are under investigation.
A hitherto "cryptic" and water-soluble EPS (named EPS-X) is elicited by elevated MnII levels
and is co-produced with curdlan. A putative EPS-X synthesis/secretion region has been
identified whose size (ca. 17 kb) suggests that EPS-X is a heterpolysaccharide. The aim of
the work is to identify the essential epx genes, determine whether EPS-X is novel
(i.e. its sub-unit composition, structure and pathway of synthesis) and whether there is
"cross-talk" between the crd and epx production systems.
(4) The regulatory cascade leading to EPS production
We hope to determine the regulatory cascade leading to curdlan (and EPS-X) production by
assessing the roles of various global regulators, namely: a two-component system (NtrBC) that
senses intracellular N-status, the "alarmone" (RelA) that initiates the bacterial stringent
response to environmental stress and the metalloprotease/chaperone (FtsH) that is essential
for survival in stationary phase. The role of additional, positive-acting, functions that may
be curdlan-specific (the CrdR regulatory protein) or EPS-X-specific (an orphan sensor kinase)
are also being studied.
(5) The biological role(s) of EPS (horses for courses?)
EPS production is a complex process initiated by an interplay of various environmental factors
(physical and nutritional) and cellular physiological triggers. The EPS armoury of
Agrobacterium is impressive - it has the capacity to produce at least six though
not simultaneously, suggesting one or more combinations of specific elicitors for each EPS.
The success of Agrobacterium as a soil saprophyte may be underscored by its EPS
versatility. We are studying the epidemiology of curdlan production by agrobacteria from
local soils and comparing the roles of cellulose and curdlan in natural contexts
(e.g. attachment to, and survival on, plant tissues; resistance to soil predators and physical
stressors e.g. temperature, dessication, toxic agents).
Key References
